A promising area of pharmaceutical intervention is the use of macromolecules that act within cells. These include gene therapy, natural and recombinant toxins, immunotoxins, and antibodies. One significant technical barrier is passing the bioactive macromolecules from the extracellular fluid (the cis-side of the membrane) through the bilipid cell membrane to the cytoplasm (the trans-side).
Generally, macromolecules enter the cell by endocytosis. Endocytosis is an ongoing process whereby the cell recycles its membrane components and internalizes molecules bound to its surface. During endocytosis, the cell membrane invaginates into the cell's interior and then pinches off to form an endosome. Endosomes comprise a complete membrane that encloses its internal contents (the cis-side) and separates them from the cytoplasm of the cell (the trans-side). After endosome formation, proton pumps within its membrane act to reduce the pH within the endosome to approximately 5.0 to 5.5. At a later stage, many endosomes merge with lysosomes where their contents are degraded.
In one mechanism of endocytosis-type cell-membrane transport, the toxins remain in endosomes that undergo processing and ultimately merges with the endoplasmic reticulum of the cell. In the endoplasmic reticulum there is a membrane transporter that translocates the molecule into the cytoplasm. This route is used by cholera toxin and the toxin ricin. This route is difficult for most complex pharmaceuticals as they are degraded during the prolonged endosomal stage.
In a second, and probably the most common, method by which toxins, viruses and pharmaceuticals with amphipathic regions (i.e., regions that contain both hydrophobic and hydrophilic amino acids) enter cells, the toxin is internalized in the endosome as described above and then passes through the endosome membrane when the interior pH becomes acidic. At acidic pH, these amphipathic regions become more hydrophobic and merge with the endosome membrane. Once incorporated in the membrane, a pore is formed through which the toxic or catalytic part of the molecule passes from the endosome into the cytoplasm.
The amphipathic bacterial toxins include the clostridial neurotoxins from Clostridia botulinum, berati, butyricum and tetani, Clostridia botulinum toxin C2, Clostridia perfringens iota toxin, Clostridia difficile B toxin, anthrax toxin, diptheria toxin, and others. All of these toxins have a basic two-component protein structure: (1) a toxic protein chain containing a catalytic domain that can perform intracellular intoxication; and (2) a protein with both a binding and translocation domain. In most toxins, the two protein components are linked covalently or tightly coupled by non-covalent forces. In others, such as Botulinum toxin C2, the two components are independent proteins, and they only interact on cell surfaces. Anthrax toxin is unusual in that the same binding/translocation protein chain, called protective antigen, can translocate either of two independent toxic proteins, lethal factor and edema factor. The binding/translocation protein chain is further subdivided into: (1) a binding domain, which recognizes one or more receptors on the surface of cells; and (2) an amphipathic domain, which can translocate the molecule through cellular or endosome membranes at acidic pH.
The binding/translocation protein chains from these wild type toxins have been separated and used as carriers to bring molecular ‘cargo’ into cells: other biological useful molecules (e.g., tetanus neurotoxin/superoxide dismutase WO/0028041A1: Delivery Of Superoxide Dismutase To Neuronal Cells, hereby incorporated herein by reference). Toxins used in this manner include botulinum and tetanus toxins, diptheria toxin, botulinum toxin C2, and anthrax toxin. The amphipathic regions have been sequenced for the translocation domains of many bacterial toxins and viruses, and based on this knowledge, novel recombinant amphipathic moieties have been produced. At present some of these natural and recombinant amphipathic proteins have been shown to capable of translocating across the endosome membrane when the endosome interior becomes acidic. However, few amphipathic protein conjugates are presently approved for clinical use.
1. Botulinum Neurotoxin
Wild type clostridial neurotoxins, specifically those from Clostridia botulinum, are amphipathic protein conjugates with unique properties that make them beneficial in medical applications. First, in their natural or wild type form, they have specificity for neurons, particularly motor neurons. Second, they can block neuromuscular transmission for extended periods, from days to months depending on the serotype. Third, in most clinical applications they have been used at doses that are below the level of immunological recognition. Fourth, as they are remarkably safe for human use when injected into local areas such as muscles because there is little systemic spread of the toxin.
The clostridial neurotoxins include seven serotypes of botulinum neurotoxins, termed A-G (A, B, C1, D, E, F, and G), and a single serotype of tetanus toxin (tetanus neurotoxin). These toxins all have a molecule size of ˜150 kD and are comprised of a heavy-chain (˜100 kD) and a light chain (˜50 kD) that are covalently linked by a disulphide bridge at their N-terminals. The heavy chain consists of the binding domain (fragment C) at the C terminal and a translocation domain (fragment B, which is the amphipathic protein) at the N-terminal end. The light chain (fragment A) is the toxic domain, however, it also contains its own small amphipathic region. These neurotoxins are exceptional due to their specific binding to neurons and their specific catalytic action on the SNARE proteins, which are involved in neurotransmission. Botulinum neurotoxins A, C, and E cleave SNAP-25, in addition botulinum neurotoxin/C cleaves syntaxin 1. botulinum neurotoxins B, D, F, G and tetanus toxin cleave VAMP-2.
The C fragments of the clostridial neurotoxins have affinity for the presynaptic membrane of neurons, and particularly the membrane of motor neurons. Clostridial neurotoxin binding is believed to involve two receptors: (1) polysialo-gangliosides accumulate clostridial neurotoxins on the plasma membrane surface, and (2) protein receptors then mediate specific endocytosis. This hypothesis was supported by the demonstration of the binding of botulinum neurotoxins B to the neuronal membrane protein synaptotagmin in the presence of GT1b, and the recent identification of GPI-anchored glycoproteins in neuronal rafts as specific receptors for the HC-fragment of tetanus neurotoxin.
After a clostridial neurotoxin binds to the presynaptic surface, it is internalized by incorporation into endosomes. When the interior of the endosome reaches about pH 5.5, the amphipathic B-fragment merges with the membrane and forms a pore that allows the light chain to pass through to the cell's cytoplasm. While passing through the membrane the disulfide bond is broken and the light chain is released into the cytoplasm and exerts its toxic effect.
The toxic action of all clostridial neurotoxin light chains is to cleave proteins necessary for attachment of internal vesicles to the cell membrane. The production and docking of these vesicles is a highly regulated process that is present in all eukaryotic cells including single-cell organisms such as yeast. The vesicle membranes merge with the cell membrane thereby adding new membrane bound proteins while simultaneously discharging the vesicle's contents into the extracellular environment. In neurons, these vesicles contain neurotransmitters and neuropeptides. Botulinum neurotoxin A and E cleaves SNAP-25; botulinum neurotoxin C cleaves SNAP-25 and syntaxin 1; and tetanus neurotoxin and botulinum neurotoxin types B, D, F and G cleave VAMP (vesicle associated membrane protein, also called synaptobrevin).
Botulinum neurotoxins A and B are the serotypes currently approved by the FDA for human use. Direct injection into extra-ocular muscles was found to be beneficial in the treatment of strabismus. Subsequently, botulinum neurotoxin A has been used to treat a variety of spastic or hyper-functional muscle disorders. Botulinum neurotoxin A has also been used for the treatment of smooth muscle hyper-function (e.g., cricopharyngeal spasm). Recently, botulinum neurotoxin has been used for treatment in connection with the cholinergic nerves of the autonomic nervous system. These uses include arresting of secretions, such as sweating and post-nasal drip.
2. Tetanus Neurotoxin
Tetanus neurotoxin exhibits fundamental differences relative to botulinum neurotoxin. First, tetanus neurotoxin binds and enters into all peripheral neurons: motor, autonomic (parasympathetic and sympathetic) and sensory neurons, including those that transmit pain signals. In contrast, botulinum neurotoxin binds and enters only motor neurons and autonomic parasympathetic neurons.
Second, at physiological doses, in contrast to botulinum neurotoxin, tetanus neurotoxin does not use the acidified endosomal pathway to enter peripheral neurons. Although internalized in the same manner as botulinum neurotoxin, the specific receptors to which tetanus neurotoxin binds allows for preferential sorting of the endosome. Tetanus-neurotoxin-containing endosomes become non-acidified vesicles that are transported retrograde to the motor neuron cell body in the central nervous system or sensory ganglia. Upon reaching the cell body, tetanus neurotoxin is released into the presynaptic space and preferentially binds to inhibitory neurons that use glycine or GABA as their neurotransmitter. When tetanus neurotoxin is taken up by inhibitory neurons in the central nervous system, it then goes through the acidified endosomal stage and acts much like that of botulinum neurotoxin in peripheral neurons. Accordingly, tetanus neurotoxin preferentially blocks inhibitory activity. The resulting unopposed excitatory activity causes muscles to contract uncontrollably, a condition called spastic paralysis. Although the clinical condition known as tetanus is a systemic intoxication, it is known that tetanus neurotoxin can also act in localized areas in mammals. At doses comparable to those that cause paralysis with botulinum neurotoxin A, tetanus neurotoxin causes a local increase in motor, autonomic and/or sensory neuron activity.
At high doses tetanus neurotoxin can cause paralysis by blocking neurotransmission both centrally and peripherally. At doses, 10 to 2000 times that needed for excitation, tetanus neurotoxin blocks both excitatory and inhibitory neurons in the central nervous system. These high doses risk local and systemic side effects. In addition, the binding domain of tetanus neurotoxin can be separated from the remainder of the molecule by digestion with the enzyme papain. Upon digestion, the resulting fragment is called tetanus neurotoxin A-B fragment and contains the light chain connected by a disulphide bridge to the translocating domain of the heavy chain. Since the A-B fragment is missing its binding fragment, it can no longer both bind and undergo retrograde transport. But the A-B fragment can cross the cell membrane and paralyze the neuromuscular synapse and cause a flaccid paralysis. But this effect requires tens of thousands more molecules of A-B fragment to than that needed for the excitation caused by wild type tetanus neurotoxin. The mechanism for this effect seems to the non-specific pinocytosis of tetanus neurotoxin A-B fragment by cells.
Finally, another unusual attribute of tetanus neurotoxin that it is internalized by some non-neuronal cell types. The most clinically useful of these are the macrophages that migrate to areas of inflammation. Tetanus neurotoxin blocks the release of inflammatory mediators and enzymes by macrophages, thereby decreasing the inflammatory response.
International Application WO 02/00172 (published Jan. 3, 2002), hereby incorporated herein by reference, teaches a wide variety of methods for using tetanus neurotoxin by increasing or decreasing neural activity or non-neural cellular activity.
The wild type amphipathic protein conjugates such as clostridial neurotoxin conjugates have wide potential as therapeutic agents. For example, the selective motor neuron binding of the neurotoxin heavy chain has been combined with the enzyme superoxide dismutase for the treatment of motor neuron degenerative diseases. The CNS transport abilities of the tetanus neurotoxin heavy chain or its He fragment are especially useful as it is one of the few vectors that can bypass the blood brain barrier (rabies and herpes virus being two others). Due to the universal nature of the vesicle docking process in cells, the use of clostridial neurotoxin light chains combined with cell-type specific amphipathic proteins holds great promise for the treatment of a wide variety of clinical conditions. Very specific targeting of cell types is plausible using recombinant technology to incorporate monoclonal immunoglobulins into amphipathic proteins. Unfortunately, however, as discussed below, introduction of these novel compounds is limited because of the inefficiencies of the endosomic process.
3. Disadvantages of Endosomic Transport of Amphipathic Protein Conjugate into Cells
Although, as discussed above, amphipathic protein conjugates—such as clostridial neurotoxins—have potential medical applications, their usefulness is limited by inefficient transport into cells. As discussed above, amphipathic protein conjugates enter cells by way of endocytosis and then require translocation across the endosome membrane. This is a disadvantage for a variety of reasons. In most cases, only a small percentage of the active moieties survive the various steps of cell binding, endocytosis, endosome acidification, and translocation. This inefficiency increases the incidence of side effects and the induction of immune reactions.
Binding of some amphipathic protein conjugates to cell membranes is highly specific to the cell type. This is an advantage for some conditions but precludes their use in other conditions where binding affinity is low. Further, the binding of the amphipathic protein conjugates to the surface of membranes prior to endocytosis can be prolonged as they await the normal cell turnover of cell membrane to reach them. Therefore, these molecules are exposed to degrading extracellular enzymes, and in some cases neutralizing antibodies. Once internalized into the cell within an endosome, acidification of the endosome to induce release of the active moiety into the cell can take hours. Accordingly, translocation across the endosome membrane is the rate-limiting step in the entry of the active moiety into the cell. Another disadvantage of amphipathic protein conjugates, is that the endosome contains its own assortment of enzymes that can degrade the conjugate. For example, during the treatment of cancer, the development of multi-drug resistance by cancer cells is believed to involve molecular changes in the endosome that cause the drug to be removed from the cell.
Therefore there is a need in the art for a method allowing amphipathic protein conjugates to bypass the endosomal stage and translocate directly across cell membranes into cytoplasm.
Basic research studies have demonstrated direct membrane translocation by way of culture mediums that mimic the acidic conditions of the endosome. In 1980, it was found that when cells in culture were exposed to diphtheria toxin in an acidic medium, the toxin would translocate directly into the cytoplasm. This advance was of basic science importance as it simplified the study of how the toxin interacts with cell membranes,. This is a much easier task then studying the toxin's interaction with the membranes of an internal organelle such as the endosome. Subsequently the ability to translocate directly into the cytoplasm has been demonstrated for a number of bacterial toxins such as anthrax toxin (lethal factor and adenylate cyclase), Clostridium botulinum C2 toxin, Clostridium difficile toxin B, Clostridia sordellii lethal toxin and the clostridial neurotoxins.
The advantages of direct translocation on the efficiency of an amphipathic protein-conjugates has been demonstrated for the Clostridium sordellii lethal toxin. When cultured cells were exposed to lethal toxin at pHs from 4.0 to 5.0 for only 10 minutes it increased the rate of intoxication over 5-fold, lowered the minimal intoxicating dose by over 100-fold, and allowed complete substrate modification within 2 h, instead of the 11 hours needed for the endosomal route.
Regarding clostridial neurotoxin, native and recombinant botulinum neurotoxin attaches to artificial bilipid layers coated with gangliosides, and translocate their light chains within seconds after exposure to pH 5 on the cis side when the trans side is held at pH 7.0. In addition, acidic cell culture medium allows clostridial neurotoxin to enter cells that have no specific binding sites. For example, botulinum neurotoxin-B has been demonstrated to translocate into cultured colon carcinoma cells and neutrophils by incubation in medium at pH 4.7. Moreover, even isolated clostridial neurotoxin light chains can translocate rapidly through bilipid membranes at pH 4.0. This effect is believed to be due to the presence of a separate amphipathic region in the light chain.
In summary, acidic medium rapidly speeds the translocation of amphipathic proteins-conjugates into cells they normally enter by the endosomal route, allows them to enter cells that they normally cannot enter, and in certain cases even allows the direct entry of the “cargo” molecule into cells.
Note that all the above-described experiments were performed to study how membrane translocation occurs or to study the intracellular effects of specific molecules and do not teach, suggest or even anticipate the use of acid mediated translocation in vivo.
In addition to amphipathic proteins, there are others possible mechanisms of protein translocation into cells. Membrane transduction proteins have recently been identified that directly bind and possibly merge with membranes and can translocate molecular cargo into cytoplasm. These proteins include part of the human immunodeficiency virus Tat, Drosophilae Antennapedia (Penetran), and Transportan (13 amino acids from galanin and wasp venom mastoporan). Based on studies of these proteins artificial membrane transduction proteins have been developed such as oligoarginine. Finally there are a variety of newer methods being studied that involve encapsulating the bioactive cargo molecule. The use of these substances in conjunction with a toxic moiety could substitute for the amphipathic moiety in all examples disclosed in this specification.
The present invention may be understood more fully by reference to the following detailed description and illustrative examples, which are intended to exemplify non-limiting embodiments of the invention.